This is a resubmission of a Phase II proposal, 2R42CA124191-02, which received a priority score of 181, to develop a tissue-based genetic test for BRCA2 hereditary ovarian cancer. Six percent of unselected US ovarian cancers are BRCA2 hereditary cancers with truncating mutations in the BRCA2 gene. It is important to know which ovarian cancer patients have BRCA2 hereditary ovarian cancer because BRCA2 patients have an increased risk of breast cancer and their relatives with mutations have an increased risk of breast and ovarian cancer. These subsequent cancers would be discovered early or prevented if widespread genetic screening was available and recent data indicates that BRCA hereditary cancers respond to targeted therapies such as cisplatinum and PARP inhibitors. We have developed an antibody-based tissue truncation method to identify BRCA2 hereditary cancers and were able to correctly identify 19/20 BRCA2 hereditary ovarian cancers and 48/50 sporadic ovarian cancers. This approach visualizes protein truncation by showing with immunohistochemistry (IHC) that the N-terminus is present but the C-terminus is absent. The product (test) will be an antibody-based IHC diagnostic kit for cancer tissue samples which hospital labs will use to identify hereditary cancers. The completed milestones for the Phase I proposal were: 1) Determine optimal tissue preparation protocols for IHC with both N-terminal and C-terminal BRCA2 antibodies. 2) Develop a quantitative scoring system for N-terminal and C-Terminal BRCA2 immunostaining on ovarian tissues with intra-assay variability of 15% and inter-assay variability of 20% or less. 3) Demonstrate that a C-terminal to N- terminal ratio for protein truncation (BRCA2 Truncation Ratio) can distinguish 20 hereditary BRCA2 ovarian cancers from 50 sporadic ovarian cases with greater than 90% sensitivity and specificity (comparing different scoring systems). 4) Develop tissue based methods for PCR-sequencing to validate results of IHC studies using the sequencing benchmark. We have completed these milestones and now propose two additional milestones for Phase II: 1a: Determine the specificity of the BRCA2 truncation ratio in 50 ovarian cancers in patients known to have BRCA1 mutations;1b: Determine the specificity of the truncation ratio in 10 ovarian cancers in patients reported to have polymorphisms/missense variants of BRCA2;2) Determine if the BRCA2 truncation test can achieve 90% sensitivity and specificity in a clinical trial of 500 ovarian cancer samples (sample size to provide 30 BRCA2 hereditary cancers). The medical application of breast/ovarian cancer genetic testing is a proven market since Myriad Genetics annually grosses $150 million on DNA sequencing. PUBLIC HEALTH RELEVANCE: Ovarian Cancer relevance: Successful completion of this Phase II research will establish a new tissue-based genetic test for BRCA2 hereditary ovarian cancer based on an IHC method to visualize protein truncation. This simpler more widely applicable approach will identify many more families with hereditary breast and ovarian cancer and consequently help patients and their relatives by identifying individuals likely to develop subsequent breast or ovarian cancer which might be prevented by tamoxifen and/or screening exams and X- rays.